Development and differentiation of B cells in health and disease plays an important role in pathomechanism of immunodeficiencies e.g. Bruton's x-linked agammaglobulinemia (XLA), autoimmune diseases e.g., systemic lupus erythematosus (SLE), and cancers e.g., B cell follicular and marginal zone lymphomas. The long-term goal of this proposal is to define B cell antigen receptor (BCR) signaling mechanisms that control the development of functional peripheral B cells. Transitional B cells go through at least two checkpoints at early (T1) and late (T2) stages of B cell ontogeny, to give rise to a functional B cell repertoire that recognizes nonself antigens. Engagement of the BCR in vitro leads to death of T1 cells, whereas under similar conditions T2 cells survive, divide, and develop into mature B lymphocytes. Thus, we hypothesize that stage-specific differences in the BCR signalosomes underlie the elimination of autoreactive T1 cells and the maturation of T2 to functional B cells. Consistent with this hypothesis, genetic evidence demonstrates that multiple BCR signaling components, including PI3K, PLC-g2, and BTK, are required for T2 to mature B cell transition, whereas T1 cells develop into T2 cells in their absence. Additionally, the BTK/PLC-g2 signaling axis mediates BCR-induced production of the lipid second messenger diacylglycerol (DAG) in T2 but not in T1 cells, suggesting a stage-specific role for DAG- dependent signals in T2 cell survival and maturation. Further, we have found, what was thought to be a T cell-specific adapter protein, SKAP55 to be preferentially expressed in T1 B cells. In Specific Aim 1, we will characterize T1- and T2-specific BCR signal transducers using cutting edge proteomics approaches. In addition, we will elucidate the signaling mechanisms of SKAP55 in T1 cell maturation in vivo. Specific Aim 2 will dissect the role of BCR-inducible phospholipases involved in phosphoinositide metabolism and the production of DAG in T1 and T2 cells. Experiments in Specific Aim 3 will assess whether transitional B cell survival and maturation in vivo depend upon DAG-mediated activation of Ras pathways. Upon completion of this project, we expect to define the stage-specific mechanisms that underlie BCR action and mediate B cell development in peripheral lymphoid organs. Further, our studies have the potential to advance our understanding of positive and negative selection within transitional B cells. This new knowledge can lead to the development of novel therapeutics for the treatment of B cell-mediated autoimmune diseases and immunodeficiencies and B cell lymphomas. [unreadable] [unreadable]